CN113242858A

CN113242858A - Preparation and application of protein receptor kinase inhibitor

Info

Publication number: CN113242858A
Application number: CN201980067895.1A
Authority: CN
Inventors: 江磊; 冯志勇; 路小丽; 尚珂; 寿建勇; 吴淡宜; 徐圆; 许灵灵; 张淑芸; 张宇星
Original assignee: Shanghai Ennovabio Pharmaceuticals Co Ltd
Current assignee: Shanghai Ennovabio Pharmaceuticals Co Ltd
Priority date: 2018-10-15
Filing date: 2019-10-15
Publication date: 2021-08-10
Also published as: CN111039947A; WO2020078360A1

Abstract

The invention provides a preparation method of a protein receptor kinase inhibitor. Specifically, the invention discloses a compound shown as a formula I, or a stereoisomer or racemate thereof, or a pharmaceutically acceptable salt thereof, wherein the definition of each group is described in the specification. The invention also discloses application of the compound as a TRK kinase inhibitor.

Description

Preparation and application of protein receptor kinase inhibitor

Technical Field

The invention relates to the technical field of medicines, and particularly relates to a protein receptor kinase inhibitor.

Background

Tropomyosin-receptor kinase (TRK) is a nerve growth factor receptor, belongs to a receptor tyrosine kinase family, mainly comprises three highly homologous members of TRKA, TRKB and TRKC, and is respectively encoded by three genes of NTRK1, NTRK2 and NTRK 3. These receptor tyrosine kinases are mainly expressed in nervous tissues and play an important role in development and physiological functions of the nervous system through activation of neurotrophic factors nts (neurotropins). TRK function as tyrosine kinase receptors, each TRK having a signaling pathway that binds to and activates its downstream ligand. NGF (nerve growth factor) specifically binds to and activates TRKA; the TRKB ligand comprises BDGF (broad-derived growth factor) and NT-4/5 (neurotropin-4/5); NT-3 specifically binds to and activates TRKC. All three TRK receptors contain an extracellular domain for ligand binding, a transmembrane domain, and an intracellular domain with kinase activity.

When a specific ligand is combined with an extracellular domain of a corresponding receptor, oligomerization of the receptor and phosphorylation of specific tyrosine residues in an intracytoplasmic kinase domain are triggered, so that downstream signal paths such as Ras/MAPK, PLC gamma/PKC and PI3K/AKT signal paths are activated, and a series of physiological processes such as proliferation, differentiation and survival of nerve cells are regulated. The TRK signaling pathway is usually precisely regulated, and its abnormal activation is closely related to tumorigenesis. Research results show that there are many mechanisms causing abnormal activation of TRK pathway, including gene fusion, protein overexpression and single nucleotide mutation, and these abnormalities are closely related to the pathogenesis of tumor, especially NTRK gene fusion has been proved to be an important factor causing multiple tumorigenesis, and is independent of the tissue source and type of tumor. Under the rapid development of current second-generation sequencing technologies and precision medicine, more and more NTRK fusion genes are found, such as ETV6-NTRK3, MPRIP-NTRK 1, CD 74-NTRK 1 and the like. The results of recent clinical trials show that the fusion genes are very effective anti-cancer targets, and the tumors containing the NTRK fusion genes have very significant response rate to TRK inhibitors.

Therefore, more and more TRK target inhibitors are reported, e.g. (WO2010048314, WO2011146336, WO 2017004342). Meanwhile, in the clinical trial stage, it has been found that some of the patients receiving the treatment have a drug resistance phenomenon and proved to be caused by partial base mutation of the enzyme activity region, such as mutation of NTRK 1G 595R or G667C, G623R or G696A of NTRK 3. Therefore, there is still a need in the art to develop a new generation of TRK kinase inhibitors to solve the above problems.

Disclosure of Invention

The invention aims to provide a novel TRK kinase inhibitor, a preparation method and application thereof.

In a first aspect of the present invention, there is provided a compound represented by formula I, or a stereoisomer or racemate thereof, or a pharmaceutically acceptable salt thereof:

wherein the content of the first and second substances,

ring a has a structure represented by the following formula:

each X⁴Each independently is C or N;

each X⁵Each independently is CR₇Or N; wherein R is₇Is hydrogen, deuterium, halogen, NH₂Or OH;

R ₁、R ₂taken together with the nitrogen atom to which they are attached to form a 6-12 membered fused ring (including bicyclic fused ring, tricyclic fused ring), bridged ring or spiro ring (including substituted or unsubstituted fused rings (including bicyclic fused ring, tricyclic fused ring), bridged ring or spiro ring); wherein said fused, bridged or spiro ring contains 1 to 3 heteroatoms selected from nitrogen atoms, oxygen atoms and sulfur atoms;

R _Ais composed of

Wherein the content of the first and second substances,

X ₁、X ₂、X ₃each independently is CR₆Or N; r₆Is hydrogen, deuterium, hydroxy, halogen, substituted or unsubstituted C1-C6 alkyl, or substituted or unsubstituted C1-C6 alkoxy;

n is 1,2 or 3;

each R is₃Each independently is hydrogen, deuterium, hydroxy, halogen, trifluoromethyl, cyano, amino, amido, substituted or unsubstituted C1-C6 alkyl, or substituted or unsubstituted C1-C6 alkoxy;

or n is 2 and two R₃co-form-O- (CH)₂) _m-O-; m is an integer of 1 to 4;

R _Bis a substituted or unsubstituted 4-7 membered heterocyclic ring (said heterocyclic ring containing 1 to 3 hetero atoms selected from the group consisting of a nitrogen atom, an oxygen atom and a sulfur atom); - (CH)₂) _m1-、-O-(CH ₂) _m2-O-; wherein m1 and m2 are each independently an integer of 1 to 4

Or R_BIs-substituted or unsubstituted-C1-C10 alkylene-L₂- (wherein, L)₂Is NH, O, S or S (O)₂) Substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, -O- (CH)₂) _m-O-; m is an integer of 1 to 4;

unless otherwise specified, "substituted" means substituted with one or more (e.g., 2, 3,4, etc.) substituents selected from the group consisting of: deuterium, halogen, C1-C6 alkoxy, halogenated C1-C6 alkyl, halogenated C1-C6 alkoxy, halogenated C3-C8 cycloalkyl, methylsulfonyl, -S (═ O)₂NH ₂Oxo (═ O), -CN, hydroxy, -NH₂Carboxy, C1-C6 amido (-C (═ O) -N (Rc)₂or-NH-C (═ O) (Rc), Rc being H or C1-C5 alkyl), C1-C6 alkyl- (C1-C6 amido),

Or a substituted or unsubstituted group selected from: C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 amino, C6-C10 aryl, 5-10 membered heteroaryl having 1-3 heteroatoms selected from N, S and O, 5-12 membered heterocyclyl having 1-3 heteroatoms selected from N, S and O, - (CH) 6 alkyl₂) -C6-C10 aryl, - (CH)₂) - (5-to 10-membered heteroaryl having 1 to 3 heteroatoms selected from N, S and O), and the substituents are selected from the group consisting of: halogen, C1-C6 alkyl, C1-C6 alkoxy, oxo, -CN, -NH₂-OH, C6-C10 aryl, C1-C6 amine, 5-10 membered heteroaryl having 1-3 heteroatoms selected from N, S and O;

the dotted line represents a chemical bond or nothing.

In another preferred embodiment, the compound of formula I has the structure shown in formula I-1 below:

in another preferred embodiment, ring a is a bicyclic 9-10 membered heteroaromatic ring; wherein the heteroaromatic ring contains 1-3 nitrogen atoms.

In another preferred embodiment, ring a is selected from the group consisting of:

in another preferred embodiment, the ring a is selected from the group consisting of:

in another preferred embodiment, R₁、R ₂Together with the nitrogen atom to which they are attached form a substituted or unsubstituted 6-12 membered bicyclic, bridged or spirocyclic ring; the ring combinationThe bridged or spiro ring contains 1-3 nitrogen atoms.

In another preferred embodiment, the fused ring is a fused ring selected from the group consisting of: 3 and 5, 4 and 5, 5 and 5, 6 and 5, 7 and 5, 6 and 6, 6 and 7.

In another preferred embodiment, the spiro ring is selected from the following group: spiro [4.5] ring, spiro [ 5.5 ] ring, spiro [ 5.6 ] ring, spiro [ 5.7 ] ring.

In another preferred embodiment, R₁、R ₂Together with the nitrogen atom to which they are attached form a substituted or unsubstituted group selected from:

in another preferred embodiment, said substitution means substitution by one or more groups selected from the group consisting of: C1-C6 alkyl, oxo, C1-C6 alkylcyano.

In another preferred embodiment, substituted

The method comprises the following steps:

in another preferred embodiment, substituted

Included

In another preferred embodiment, substituted

Included

In another preferred embodiment, substituted

Included

In another preferred embodiment, substituted

Included

In another preferred embodiment, substituted

Included

In another preferred embodiment, R_ASelected from the group consisting of:

in each formula, X₁、X ₂The definition is the same as the previous definition;

on the aromatic ring, n is 1,2 or 3; and R is₃Is hydrogen, deuterium, hydroxy, halogen, substituted or unsubstituted C1-C6 alkyl, or substituted or unsubstituted C1-C6 alkoxy; or n is 2 and two R₃co-form-O- (CH)₂) _m-O-; m is an integer of 1 to 4;

r elsewhere₃、R ₄、R ₅Each independently hydrogen, deuterium, hydroxy, halogen, substituted or unsubstituted C1-C6 alkyl, or substituted or unsubstituted C1-C6 alkoxy.

In another preferred embodiment, R_AIs composed of

Wherein, X₁Is CR₆Or N; x₂、X ₃Each independently is CR₆；

n is 1,2 or 3; and R is₃Is hydrogen, deuterium, hydroxy, halogen, substituted or unsubstituted C1-C6 alkyl, or substituted or unsubstituted C1-C6 alkoxy; (ii) a Or n is 2 and two R₃co-form-O- (CH)₂) _m-O-; m is an integer of 1 to 4.

In another preferred embodiment, the compound of formula I is selected from the group consisting of:

in a second aspect of the invention, there is provided a pharmaceutical composition comprising a therapeutically effective amount of a compound according to the first aspect of the invention or a stereoisomer or racemate thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

In a third aspect of the present invention, there is provided a use of the compound according to the first aspect of the present invention or a stereoisomer or racemate thereof, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to the second aspect of the present invention, in a medicament for preventing and treating a TRK-associated disease.

In another preferred embodiment, the TRK-associated disease is selected from the group consisting of: cancer, proliferative diseases, pain, skin diseases or conditions, metabolic diseases, muscular diseases, neurological diseases, autoimmune diseases, dermatitis-induced pruritus, inflammation-related diseases, bone-related diseases.

In another preferred embodiment, the cancer is selected from cancers associated with abnormal TRK function (abnormal TRK activation due to amplification, overexpression, mutation, or gene fusion), including but not limited to: neuroblastoma, prostate cancer, thyroid cancer, lung cancer, ovarian cancer, pancreatic cancer, colorectal cancer, non-small cell lung cancer, fibrosarcoma, and the like.

In a fourth aspect of the present invention, there is provided a use of a compound according to the first aspect of the present invention or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a pharmaceutical composition according to the second aspect of the present invention, for the preparation of a pharmaceutical composition for the prevention and/or treatment of a disease associated with abnormal TRK function (abnormal TRK activation due to amplification, or overexpression, or mutation, or gene fusion).

In another preferred embodiment, the pharmaceutical composition may be administered in combination with other therapeutic agents.

In another preferred embodiment, the drug combination includes, but is not limited to, other target kinase inhibitors, immunomodulators (tumor immune checkpoint inhibitors), cell division blockers and other conventional first-line chemotherapeutic drugs.

In another preferred embodiment, the disease is selected from the group consisting of: cancer, proliferative diseases, pain, skin diseases or conditions, metabolic diseases, muscular diseases, neurological diseases, autoimmune diseases, dermatitis-induced pruritus.

In a fifth aspect of the invention, there is provided a TRK inhibitor comprising a compound according to the first aspect of the invention, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof.

In another preferred embodiment, the TRK inhibitor selectively inhibits one or more TRK kinases selected from the group consisting of: TRKA, TRKB or TRKC.

In a sixth aspect of the present invention, there is provided a process for the preparation of a compound according to the first aspect of the present invention or a stereoisomer or racemate thereof, or a pharmaceutically acceptable salt thereof, comprising the steps of:

reacting a compound of formula Ia with a compound of formula Ib in an inert solvent, thereby forming a compound of formula I;

wherein R is₁Is C1-C6 alkyl; the remaining groups are as defined for the first aspect of the invention.

In another preferred embodiment, when the compound of formula Ib further comprises a protecting group (e.g., an amino protecting group or a hydroxyl protecting group), this step further comprises a deprotection step.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Detailed Description

The inventor discovers a novel and effective TRK kinase small molecule inhibitor for the first time through extensive and intensive research. The present invention has been completed based on this finding.

Term(s) for

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the term "comprising" or "includes" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….

In this application, the term "alkyl" as a group or part of another group refers to a fully saturated straight or branched hydrocarbon chain radical consisting of only carbon and hydrogen atoms, having, for example, from 1 to 12 (preferably from 1 to 8, more preferably from 1 to 6) carbon atoms, and attached to the rest of the molecule by a single bond, including, for example, but not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2-dimethylpropyl, n-hexyl, heptyl, 2-methylhexyl, 3-methylhexyl, octyl, nonyl, decyl, and the like. For the purposes of the present invention, the term "C1-C6 alkyl" refers to alkyl groups containing from 1 to 6 carbon atoms.

In this application, the term "6-to 10-membered aromatic ring" as a group or as part of another group means an aromatic ring having 6 to 10 ring atoms, which are carbon atoms. The aromatic ring may be monocyclic or bicyclic. Such as benzene rings, naphthalene rings, and the like.

In this application, the term "5-to 10-membered heteroaromatic ring" as a group or as part of another group means a heteroaromatic ring having 5 to 10 ring atoms, at least 1 (which may be 1,2 or 3) of which is a heteroatom selected from nitrogen, oxygen and sulfur. The heteroaryl ring may be monocyclic or bicyclic. For example, a pyrimidopyrazole ring, a pyrazinoimidazole ring, a pyridopyrazole ring, a pyridoimidazole ring, a pyridopyrimidine ring, a pyridopyridine ring.

In this application, the term "6-11 membered bridged or spiro ring" as a group or as part of another group means a bicyclic ring having 6-11 ring atoms, and wherein the two rings are bridged or spiro-linked. The bridged or spiro ring contains 1 to 3 heteroatoms selected from nitrogen atoms, oxygen atoms, and sulfur atoms. For example,

compounds of the invention

The compound is a compound shown in a formula I, or a stereoisomer or racemate thereof, or pharmaceutically acceptable salt thereof.

The compounds of the present invention may contain one or more chiral carbon atoms and may therefore give rise to enantiomers, diastereomers and other stereoisomeric forms. Each chiral carbon atom may be defined as (R) -or (S) -, based on stereochemistry. The present invention is intended to include all possible isomers, as well as racemates and optically pure forms thereof. The compounds of the invention may be prepared by selecting as starting materials or intermediates racemates, diastereomers or enantiomers. Optically active isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, e.g., crystallization and chiral chromatography.

Conventional techniques for the preparation/separation of individual isomers include Chiral synthesis from suitable optically pure precursors, or resolution of racemates (or racemates of salts or derivatives) using, for example, Chiral high performance liquid chromatography, as described, for example, in Gerald Gubitz and Martin G.Schmid (Eds.), Chiral Separations, Methods and Protocols, Methods in Molecular Biology, Vol.243, 2004; m. Stalcup, Chiral Separations, Annu. Rev. anal. chem.3:341-63, 2010; fumiss et al (eds.), VOGEL' S ENCYCOPEDIA OF PRACTICAL ORGANIC CHEMISTRY 5. TH ED., Longman Scientific and Technical Ltd., Essex,1991, 809-816; heller, acc, chem, res, 1990,23,128.

The term "pharmaceutically acceptable salts" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

"pharmaceutically acceptable acid addition salts" refers to salts with inorganic or organic acids which retain the biological effectiveness of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochloride, hydrobromide, sulfate, nitrate, phosphate, and the like; organic acid salts include, but are not limited to, formates, acetates, 2-dichloroacetates, trifluoroacetates, propionates, caproates, caprylates, caprates, undecylenates, glycolates, gluconates, lactates, sebacates, adipates, glutarates, malonates, oxalates, maleates, succinates, fumarates, tartrates, citrates, palmitates, stearates, oleates, cinnamates, laurates, malates, glutamates, pyroglutamates, aspartates, benzoates, methanesulfonates, benzenesulfonates, p-toluenesulfonates, alginates, ascorbates, salicylates, 4-aminosalicylates, napadisylates, and the like. These salts can be prepared by methods known in the art.

"pharmaceutically acceptable base addition salts" refers to salts with inorganic or organic bases which maintain the biological effectiveness of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. Preferred inorganic salts are ammonium, sodium, potassium, calcium and magnesium salts. Salts derived from organic bases include, but are not limited to, the following: primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be prepared by methods known in the art.

Preparation method

The following reaction schemes illustrate methods for preparing compounds of formula I, or stereoisomers or racemates thereof, or pharmaceutically acceptable salts thereof, wherein each group is as described above. It is understood that in the following reaction schemes, combinations of substituents and/or variables in the general formulae are permissible only if such combinations result in stable compounds. It will also be appreciated that other general formulae may be prepared by methods disclosed herein (by applying appropriately substituted starting materials and modifying the synthesis parameters as required using methods well known to those skilled in the art) or known methods by those skilled in the art of organic chemistry.

The method comprises the following steps:

wherein R is₁Is C1-C6 alkyl; ring B is as defined for the first aspect of the invention.

It will also be appreciated by those skilled in the art that in the processes described below, the functional groups of the intermediate compounds may need to be protected by suitable protecting groups. Such functional groups include hydroxyl, amino, mercapto and carboxylic acid. Suitable hydroxy protecting groups include trialkylsilyl or diarylalkylsilyl groups (e.g.tert-butyldimethylsilyl, tert-butyldiphenylsilyl or trimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitable protecting groups for amino, amidino and guanidino include t-butyloxycarbonyl, benzyloxycarbonyl and the like. Suitable thiol protecting groups include-C (O) -R "(where R" is alkyl, aryl or aralkyl), p-methoxybenzyl, trityl and the like. Suitable carboxyl protecting groups include alkyl, aryl or aralkyl esters.

Protecting groups may be introduced and removed according to standard techniques known to those skilled in the art and as described herein. The use of protecting Groups is described in detail in Greene, T.W. and P.G.M.Wuts, Protective Groups in organic Synthesis, (1999),4th Ed., Wiley. The protecting group may also be a polymeric resin.

Applications of

The compound of the present invention has excellent TRK (e.g., TRKA, TRKB, TRKC) inhibitory activity, and thus the compound of the present invention and various crystalline forms, pharmaceutically acceptable inorganic or organic salts, hydrates, or solvates thereof, and a pharmaceutical composition containing the compound of the present invention as a main active ingredient may be used for the prevention and/or treatment of diseases associated with TRK kinase activity or expression amount (e.g., cancer).

In the present application, the term "pharmaceutical composition" refers to a formulation of a compound of the present invention with a vehicle generally accepted in the art for delivery of biologically active compounds to a mammal (e.g., a human). The medium includes a pharmaceutically acceptable carrier. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of active ingredients and exert biological activity.

In the present application, the term "pharmaceutically acceptable" refers to a substance (e.g., carrier or diluent) that does not affect the biological activity or properties of the compounds of the present invention and is relatively non-toxic, i.e., the substance can be administered to an individual without causing an adverse biological response or interacting in an adverse manner with any of the components contained in the composition.

In this application, the term "pharmaceutically acceptable excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye/colorant, flavoring agent, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifying agent that is approved by the relevant governmental regulatory agency for human or livestock use.

In the present application, the term "tumor" includes, but is not limited to, glioma, sarcoma, melanoma, arthroscleroma, cholangioma, leukemia, gastrointestinal stromal tumor, histiocytic lymphoma, non-small cell lung cancer, pancreatic cancer, lung squamous cancer, lung adenocarcinoma, breast cancer, prostate cancer, liver cancer, skin cancer, epithelial cell cancer, cervical cancer, ovarian cancer, intestinal cancer, nasopharyngeal cancer, brain cancer, bone cancer, esophageal cancer, melanoma, renal cancer, oral cancer, and the like.

In this application, the terms "preventing", "prevention" and "prevention" include reducing the likelihood of occurrence or worsening of a disease or disorder in a subject.

In the present application, the term "treatment" and other similar synonyms include the following meanings:

(i) preventing the occurrence of a disease or condition in a mammal, particularly when such mammal is susceptible to the disease or condition, but has not been diagnosed as having the disease or condition;

(ii) inhibiting the disease or disorder, i.e., arresting its development;

(iii) alleviating the disease or condition, i.e., causing regression of the state of the disease or condition; or

(iv) Alleviating the symptoms caused by the disease or disorder.

In the present application, the term "effective amount", "therapeutically effective amount" or "pharmaceutically effective amount" refers to an amount of at least one agent or compound that is sufficient to alleviate one or more symptoms of the disease or condition being treated to some extent after administration. The result may be a reduction and/or alleviation of signs, symptoms, or causes, or any other desired change in a biological system. For example, an "effective amount" for treatment is the amount of a composition comprising a compound disclosed herein that is clinically necessary to provide a significant remission effect of the condition. An effective amount suitable in any individual case can be determined using techniques such as a dose escalation assay.

In this application, the terms "administering", "administration", "administering", and the like refer to a method capable of delivering a compound or composition to a desired site for biological action. These methods include, but are not limited to, oral routes, via the duodenal route, parenteral injection (including intravenous, subcutaneous, intraperitoneal, intramuscular, intraarterial injection or infusion), topical administration, and rectal administration. Administration techniques useful for The compounds and methods described herein are well known to those skilled in The art, for example, in Goodman and Gilman, The pharmaceutical Basis of Therapeutics, current ed.; pergamon; and Remington's, Pharmaceutical Sciences (current edition), Mack Publishing Co., Easton, Pa. In preferred embodiments, the compounds and compositions discussed herein are administered orally.

In the present application, the terms "drug combination", "administering other therapy", "administering other therapeutic agent" and the like refer to a drug treatment obtained by mixing or combining more than one active ingredient, which includes both fixed and unfixed combinations of active ingredients. The term "fixed combination" refers to the simultaneous administration of at least one compound described herein and at least one co-agent to a patient in the form of a single entity or a single dosage form. The term "non-fixed combination" refers to the simultaneous administration, concomitant administration, or sequential administration at variable intervals of at least one compound described herein and at least one synergistic formulation to a patient as separate entities. These also apply to cocktail therapy, for example the administration of three or more active ingredients.

The main advantages of the invention are:

the compound can effectively inhibit the proliferation of KM12-LUC cells.

The compounds of the invention are capable of effectively inhibiting the phosphorylation levels of TRKA, TRKB and TRKC at the cellular level.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

The test materials and reagents used in the following examples are commercially available without specific reference.

Synthesis of intermediate 1

(R, E) -N- (2, 5-difluorobenzylidene) -2-methylpropane-2-sulfinamide

2, 5-difluorophenyl (formaldehyde) (5g,35.2mmol) and (R) -2-methylpropane-2-sulfinamide (4.47g,36.9mmol) were dissolved in dichloromethane (50ml), cesium carbonate (8.0g,24.6mmol) was added at room temperature, and then the reaction was warmed to 50 ℃ for 3h, TLC showed completion of the reaction, filtration, the filter cake was washed with dichloromethane, the filtrate was washed with brine, Na₂SO ₄Drying and spin-drying to obtain (R, E) -N- (2, 5-difluorobenzylidene) -2-methylpropane-2-sulfinamide as a yellow oily liquid (9 g).

(R) -N- ((R) -1- (2, 5-difluorophenyl) -3- (1, 3-dioxan-2-yl) propyl) -2-methylpropane-2-sulfinamide

Magnesium turnings (2g,83.3mmol) were dissolved in tetrahydrofuran (72ml) and, under nitrogen, Dibal-H (0.1ml,1.5M,0.15mmol) was added dropwise to the system at 40 ℃ for 0.5H at 40 ℃ and then a solution of 2- (2-bromoethyl) -1, 3-dioxane (14.3g,73.47mmol) in tetrahydrofuran (40ml) was slowly added dropwise to the system with temperature controlled at 40-50 ℃ and after dropping was kept at 40 ℃ for 1H. Heating was removed, the reaction was cooled to-30 ℃ and then (R, E) -N- (2, 5-difluorobenzylidene) -2-methylpropane-2-sulfinamide (9g,36.73mmol) in tetrahydrofuran (40ml) was added dropwise to the system, the temperature was controlled at-30 ℃ to 20 ℃, after completion of the addition, the mixture was stirred at-30 ℃ for 2h, TLC showed completion of the reaction, quenched with 10% aqueous citric acid and controlled at 10 ℃, extracted with dichloromethane, the organic phase was washed with saturated brine, Na₂SO ₄Drying and spin-drying gave (R) -N- ((R) -1- (2, 5-difluorophenyl) -3- (1, 3-dioxan-2-yl) propyl) -2-methylpropane-2-sulfinamide as a colorless oily liquid (15.8 g).

(R) -2- (2, 5-difluorophenyl) pyrrolidine

(R) -N- ((R) -1- (2, 5-difluorophenyl) -3- (1, 3-dioxan-2-yl) propyl) -2-methylpropane-2-sulfinamide (15.8g,43.76mmol) was added to a mixed solution of trifluoroacetic acid (32ml) and water (8ml) at room temperature, stirred at room temperature for 1h, then trifluoroacetic acid (60ml) was added to the system, triethylsilane (15.2g,131.1mmol) was added dropwise to the system, the reaction was allowed to stand overnight at room temperature, LCMS monitored for completion of the reaction, most of the trifluoroacetic acid was spun off, the residue was dissolved in hydrochloric acid (1N,100ml) and stirred for 0.5h, extracted with methyl tert-butyl ether, the organic phase was washed with hydrochloric acid (1N,50ml), the aqueous phases were combined, adjusted to pH 11 with 40% aqueous sodium hydroxide solution and then extracted with dichloromethane, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate and spin-dried to give (R) -2- (2, 5-difluorophenyl) pyrrolidine as an oily liquid (6.7 g).

Pyrazolo [1,5-a ] pyrimidin-5 (4H) -ones

Dissolving 1H-pyrazol-5-amine (20 g, 0.24 mol) and 1, 3-dimethylpyrimidine-2, 4(1H,3H) -diketone (35 g, 0.25 mol) in ethanol (200 ml), reducing the temperature of sodium ethoxide (34 g, 0.50 mol) at room temperature, adding the mixture into a reaction system in batches, heating the mixture to 70 ℃ for reaction for 3 hours, monitoring the reaction by LCMS (liquid crystal display system), cooling the reaction system to room temperature, filtering, washing a filter cake by ethanol, washing the filter cake by n-hexane, and drying to obtain pyrazolo [1,5-a ] pyrimidin-5 (4H) -one and a yellow solid (36g, yield: 94%).

3-nitropyrazolo [1,5-a ] pyrimidin-5 (4H) -ones

Pyrazolo [1,5-a ] pyrimidin-5 (4H) -one (30 g, 191.1 mol) was added in portions to nitric acid (68%) (150 ml) at room temperature, heated to 80 ℃ for reaction for 3 hours, and LCMS monitored for completion of the reaction, after the reaction system was cooled to room temperature, filtered, the filter cake was washed with water, washed with methanol, and dried to give 3-nitropyrazolo [1,5-a ] pyrimidin-5 (4H) -one as a red solid (28g, yield: 82%).

5-chloro-3-nitropyrazolo [1,5-a ] pyrimidines

Reacting 3-nitropyrazolo [1,5-a ]]Pyrimidin-5 (4H) -one (15g, 83.3mmol) and 2, 6-lutidine (17.8 g, 166.7 mmol) were dissolved in acetonitrile (150 mL) and POCl was added at room temperature₃(51 g, 333.2 mmol) is added into the system dropwise, then the temperature is raised to 80 ℃ and the mixture is stirred overnight, LCMS monitors that the reaction is finished, the solvent is spun off, the rest is dissolved in dichloromethane (200 ml), the organic phase is washed by water, saturated brine is washed, anhydrous sodium sulfate is dried, and the 5-chloro-3-nitropyrazolo [1,5-a ] is obtained by spin-drying]Pyrimidine, grey solid (15g, yield: 91%).

(R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitropyrazolo [1,5-a ] pyrimidine

5-chloro-3-nitropyrazolo [1,5-a ] pyrimidine (2.38 g, 12.02 mmol) and (R) -2- (2, 5-difluorophenyl) pyrrolidine (4 g, 12.62 mmol) were dissolved in ethanol (40ml) and tetrahydrofuran (10 ml), triethylamine (4.2 g, 42 mmol) was added at room temperature, the reaction was heated to 50 ℃ for 2 hours, the reaction was monitored by LCMS, the solvent was removed by spinning off, the residue was dissolved in dichloromethane, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and spin-dried to give (R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitropyrazolo [1,5-a ] pyrimidine as a yellow solid (3.7g, yield: 90%).

(R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-amine

Dissolving (R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-nitropyrazolo [1,5-a ] pyrimidine (3.7g, 10.72 mmol) in a mixed solution of dichloromethane (50ml), methanol (50ml) and saturated ammonium chloride solution (50ml), adding zinc powder (6.8 g, 107.2 mmol) at room temperature, reacting overnight at room temperature, monitoring the completion of the reaction by LCMS, filtering, washing the filtrate with saturated brine, drying with anhydrous sodium sulfate, spin-drying, passing through a column (dichloromethane/methanol ═ 150/1-50/1) to obtain (R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-amine, yellow solid (3.2g, yield: 94%)

¹H NMR(400MHz,CDCl3):δ(ppm)8.06(d,J＝7.6Hz,1H),7.62(s,1H),7.06-7.00(m,1H),6.91-6.87(m,1H),6.77-6.73(m,1H),5.88(d,J＝1.6Hz,1H),5.32-5.29(m,1H),3.92(d,J＝7.2Hz,1H),3.75(d,J＝7.2Hz,1H),2.78(s,2H),2.48-2.44(m,1H),2.08-2.01(m,3H).

¹⁹F NMR(400MHz,CDCl3):δ(ppm)-124.30,-124.35.

Synthesis of intermediate 2

1) Synthesis of Compounds 2-3

In a single neck flask (100mL), compound 1-1(0.58g, 2.0mmol) and compound 1-2(0.70g, 4.0mmol) were dissolved in EtOH (10.0mL) followed by the addition of AcOH (0.24g, 4.0 mmol). The reaction was stirred at room temperature for 3 hours. Then, add NaBH₃CN (0.50g, 8.0 mmol). The resulting reaction solution was stirred at 75 ℃ for 17 hours. Under reduced pressure willThe reaction solution was concentrated to dryness, and the resulting residue was dissolved in 30mL of methylene chloride and 30mL of a saturated ammonium chloride solution. After separation of the organic phase, the aqueous phase is extracted twice with dichloromethane. The combined organic phases were concentrated to dryness and the residue was chromatographed on petroleum ether and ethyl acetate to give 1-3 as a colorless oil (0.72g, 80% yield). LC-MS (ESI +) M/z [ M + H ]]450.32

2) Synthesis of Compounds 2 to 4

In a single-necked flask (50mL), compound 1-3(0.72g, 1.6mmol), CH was added₂Cl ₂(8.0mL) and TFA (1.1g, 9.6 mmol). The reaction was stirred at room temperature for 17 hours. The reaction was concentrated to dryness and the resulting crude product 1-4(1.2g, over 100% yield) was used directly in the next reaction. LC-MS (ESI +) M/z [ M + H ]]350.20

3) Synthesis of Compounds 2 to 5

In a single neck flask (100mL) were added compounds 1-4(0.49g, 1.4mmol), MeOH (28mL) and NaOMe in methanol (5.4 mol/L,0.39mL, 2.1 mmol). The reaction solution was stirred at 80 ℃ for 3 hours. The reaction solution was concentrated to dryness, and the residue was dissolved in 20mL of methylene chloride and 20mL of water. After separation of the organic phase, the aqueous phase is extracted twice with dichloromethane. The combined organic phases were concentrated to dryness to give crude 1-5(0.37g, 87% yield). LC-MS (ESI +) M/z [ M + Na ]326.14

4) Synthesis of Compound 2

In a single-neck flask (50mL), compound 1-5(0.37g, 1.24mmol), Boc was added₂O(0.35g，1.61mmol)，Et ₃N (0.13g, 1.24mmol) and CH₂Cl ₂(2.5 mL). The reaction solution was stirred at room temperature for 12 hours. The reaction was concentrated to dryness and the resulting residue was subjected to column chromatography using petroleum ether and ethyl acetate to give 1-6(0.33g, 66% yield) as a yellow oil. LC-MS (ESI +) M/z [ M + Na]426.36

Synthesis of intermediate Compound 3

1) Synthesis of Compound 3-2

Compound 1(630mg, 2.69mmol), compound 2(698mg, 4.03mmol) and methanol (15mL) were added to a single-necked flask (100mL) and reacted at room temperature for 1 hour, and sodium cyanoborohydride (253mg, 4.03mmol) was added and reacted at room temperature for 16 hours. The reaction was spin dried and the column was washed with dichloromethane and methanol to give 3 as a yellow oil (960mg, 91% yield). LC-MS (ESI) M/z (M + H)⁺392.31。

2) Synthesis of Compound 3-3

Compound 3(960mg, 2.45mmol), dioxane hydrochloride (15mL,60mmol) was added to a single-neck flask (100mL) and the reaction was allowed to react at room temperature for 2 hours. The reaction mixture was spin-dried to give 4 as a yellow oil (980mg, 100% yield). LC-MS (ESI) M/z (M + H)⁺292.24.

3) Synthesis of Compound 3

Compound 4(980mg, 2.45mmol), potassium carbonate (1.014g, 7.35mmol), and methanol (15mL) were added to a single-neck flask (100mL) and the reaction was refluxed at 75 ℃ for 3 hours. The reaction was cooled to room temperature, filtered, the filtrate was spin-dried, and the residue was purified by column chromatography using dichloromethane and methanol to give 5(390mg, yield 61%) as a yellow oil. LC-MS (ESI) M/z (M + H)⁺260.24。1H NMR(400MHz,DMSO)δ7.26(m,5H),3.59(m,2H),3.22(m,2H),2.80(m,5H),2.65(dd,J＝24.8,9.1Hz,2H),2.40(dd,J＝15.9,7.9Hz,1H),2.18(ddd,J＝12.4,7.8,4.6Hz,1H),1.67(dt,J＝12.4,7.3Hz,1H).

EXAMPLE 1N- (5- ((R) -2- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -1, 7-diazaspiro [4.4] nonane-7-carboxamide

To a solution of (R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-amine (40 mg, 0.126 mmol) in dichloromethane (4 ml) was added CDI (41 mg, 0.253 mmol), stirred at room temperature for 2 hours, followed by addition of tert-butyl 1, 7-diazaspiro [4.4] nonane-1-carboxylate (143 mg, 0.632 mmol) and N, N-diisopropylethylamine (163 mg, 1.265 mmol) in dichloromethane (2ml), the reaction was stirred for 2 hours, monitoring of liquid quality showed the formation of the product, concentrated, a hydrochloric acid/1-4 dioxane solution (4 ml, 4N) was added, stirred overnight, concentrated, and the residue was purified by preparative hplc to give the desired product (27.7 mg, 62%) as a yellow solid.

MS-ESI:m/z 468[M+H]+.

¹H NMR(400MHz,CD ₃OD)δ(ppm)8.24(s,1H),7.87(s,1H),7.16-7.07(m,1H),7.02-6.93(m,1H),6.84-6.80(m,1H),6.40-6.10(m,1H),5.41(s,1H),4.59-4.57(m,1H),4.01-3.91(m,1H),3.80-3.66(m,1H),3.62-3.50(m,2H),3.45-3.32(m,2H),3.05–2.82(m,2H),2.55-2.40(m,1H),2.12-1.96(m,4H),1.94–1.72(m,4H).

Example 27-amino-N- (5- ((R) -2- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -2-azaspiro [4.5] decane-2-carboxamide

N- (5- ((R) -2- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -7-carbonyl-2-azaspiro [4.5] decane-2-carboxamide (30 mg, 0.06 mmol), ammonium acetate (23 mg, 0.30 mmol) were dissolved in methanol (4 ml), stirred at room temperature for 2 hours, sodium cyanoborohydride (11 mg, 0.18 mmol) was added, stirred at room temperature for 2 hours, concentrated, and the residue was purified by preparative high performance liquid chromatography to give the desired product (27.7 mg, 62%) as a yellow solid.

MS-ESI:m/z 496[M+H]+.

¹H NMR(400MHz,CD ₃OD)δ(ppm)8.25(d,J＝4.0Hz,1H),7.87(s,1H),7.14-7.08(m,1H),7.01-6.93(m,1H),6.84-6.80(m,1H),6.15(brs,1H),5.41(s,1H),4.00-3.91(m,1H),3.75-3.73(m,1H),3.49-3.45(m,2H),3.22-3.15(m,1H),2.88-2.77(m,1H),2.51-2.41(m,1H),2.07-1.11(m,16H).

EXAMPLE 3N- (5- ((R) -2- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -1, 4-dimethyl-5-carbonyloctahydropyrrolo [3,4-e ] [1,4] diazepine-7 (1H) -carboxamide

N- (5- ((R) -2- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -4-methyl-5-carbonyloctahydropyrrolo [3,4-e ] [1,4] diazepin-7 (1H) -carboxamide (14 mg, 0.027 mmol), aqueous formaldehyde (1 ml) and acetic acid (2 drops) were dissolved in methanol (3 ml), stirred at room temperature for 2 hours, sodium cyanoborohydride (5 mg, 0.082 mmol) was added, stirred at room temperature for 1 hour, concentrated, and the residue was purified by preparative high performance liquid chromatography to give the desired product (27.7 mg, 62%) as a yellow solid.

MS-ESI:m/z 525[M+H]+.

¹H NMR(400MHz,CD ₃OD)δ(ppm)8.25(d,J＝6.4Hz,1H),7.87(d,J＝4.8Hz, 1H),7.16-7.10(m,1H),6.99-6.93(m,1H),6.84-6.79(m,1H),6.14(brs,1H),5.39(brs,1H),4.00-3.87(m,4H),3.80-3.71(m,2H),3.45-3.33(m,2H),3.01(s,3H),3.01-2.95(m,2H),2.67-2.59(m,1H),2.49-2.36(m,2H),2.32(s,3H),2.07-1.96(m,3H).

The corresponding starting materials were replaced by analogous conditions to those of examples 1 to 3, giving the compounds shown in the following table:

comparative example 12- (1- (6- ((R) -2- (2, 5-difluorophenyl) pyrrolidin-1-yl) imidazo [1,2-b ] pyridazine-3-carbonyl) -3-hydroxypyrrolidin-3-yl) -N-methylacetamide

Preparation of the title Compound under analogous conditions to example 1

MS(ESI):m/z＝485[M+H]+.

1H NMR(400MHz,CD3OD)δ(ppm)7.75-7.70(m,1H),7.65(t,J＝5.5Hz,1H),7.18–7.04(m,1H),7.04–6.78(m,3H),5.38-5.32(m,1H),4.08–3.43(m,6H),2.78–2.60(m,3H),2.58-2.40(m,2H),2.35-2.15(m,1H),2.14–1.69(m,5H).

Comparative example 2 (6- ((R) -2- (2, 5-difluorophenyl) pyrrolidin-1-yl) imidazo [1,2-b ] pyridazin-3-yl) (3- ((dimethylamino) methyl) -4-hydroxypyrrolidin-1-yl) methanone

Preparation of the title Compound under analogous conditions to example 1

MS(ESI):m/z＝471[M+H]+.

1H NMR(400MHz,CD3OD)δ(ppm)7.77-7.70(m,1H),7.67-7.65(m,1H),7.18–7.08(m,1H),7.00-6.80(m,2H),5.34(t,J＝9.4Hz,1H),4.56(s,1H),4.15-4.05(m,1H),4.00-3.63(m,4H),3.60-3.45(m,1H),3.44–3.33(m,1H),3.15-2.88(m,1H),2.56–2.40(m,2H),2.39–1.92(m,9H).

Biological example 1: TRKA, TRKB, TRKC kinase in vitro activity assay

Experimental Material

Recombinant human TRKA, TRKB, TRKC proteins were purchased from Carna Biosciences. HTRF kinEASE TK kit was purchased from CisbioBioassays. The BioTek microplate reader Synergy Neo 2 plate was used.

Experimental methods

Test compounds were diluted in 3-fold concentration gradient to a final concentration of 1 μ M to 0.05nM 10 concentrations, two replicate wells per concentration; the content of DMSO in the assay reaction was 1%.

TRKA enzyme reaction:

0.2 ng/. mu.l TRKA protein kinase, 1. mu.M TK Substrate-biotin (Substrate-biotin) polypeptide Substrate, 14.68. mu.M ATP, 1 Xenzyme buffer, 5mM MgCl₂1mM DTT. The detection plate is White Proxiplate384-Plus plate (PerkinElmer), the reaction is carried out for 40 minutes at room temperature, and the reaction system is 10 mu l.

TRKB enzyme reaction:

0.037 ng/. mu.l TRKB protein kinase, 1. mu.M TK Substrate-biotin (Substrate-biotin) polypeptide Substrate, 4.77. mu.M ATP, 1 Xenzyme buffer, 5mM MgCl₂，1mMMnCl ₂1mM DTT. The assay plate was a White Proxiplate384-Plus plate (PerkinElmer) and reacted at room temperature for 50 minutes in a 10. mu.l reaction system.

TRKC enzyme reaction:

0.037ng/μl TRKprotein C kinase, 1. mu.M TK Substrate-biotin (Substrate-biotin) polypeptide Substrate, 25.64. mu.M ATP, 1 Xenzyme buffer, 5mM MgCl₂1mM DTT. The detection plate is White Proxiplate384-Plus plate (PerkinElmer), the reaction is carried out for 40 minutes at room temperature, and the reaction system is 10 mu l.

Reaction detection:

mu.l of detection reagent were added to the reaction plate, containing SA-XL665 at a final concentration of 0.125. mu.M and 1 XTK-antibody 5. mu.l, incubated overnight at room temperature, Synergy Neo 2 read plate.

Data analysis

The 665/620Ratio value was converted into an inhibition Ratio (%) (1-Ratio) by the following equation_test/Ratio _max)×100％。Ratio _maxRatio as a positive control without test Compound_testThe values were measured for each concentration of different compounds. IC50(nM) data were obtained by 4-parameter curve fitting, as shown in Table 1.

TABLE 1

Compound (I)	TRKA(nm)	TRKB(nm)	TRKC(nm)
Example 1	++	+	++
Example 2	+++	+++	+++
Example 3	+++	+++	+++
Example 4	++	++	++
Example 5	+++	++	+++
Example 6	+++	++	+++
Example 7	++	+	++
Example 8	++	++	++
Example 9	+++	+++	+++
Comparative example 1	+	-	-
Comparative example 2	-	-	-

Wherein, + + + + denotes IC₅₀< 10nm, + denotes 10nm < IC₅₀< 100nm, + denotes 100nm < IC₅₀< 500nm, -represents IC₅₀＞500nm

Bioassay example 2 detection of cytological level of TRK kinase Activity by the Elisa method

NIH-3T3 cell line stably expressing normal TRKA or TRKB or TRKC was constructed by plasmid transfection.

Cells were seeded on the first day in 96-well cell culture plates at 10000 cells/well in normal medium (DMEM + 10% FBS). The next day, the medium containing 0.5% FBS was starved overnight. On the third day, cells were treated with different concentrations of test compound for 1 hour and then stimulated with 100ng/ul growth factor for 10 minutes (NGF for TRKA, BDNF for TRKB, NT-3 for TRKC). Placing the cell culture plate on ice; the supernatant was removed and rinsed once with pre-cooled PBS. The lysis buffer (cellsingailing Technology) attached to the elisa kit was diluted with double distilled water and protease and phosphatase inhibitors were added. The prepared cell lysate was added to the well plate and allowed to stand on ice for 20 minutes. The cell lysate was blown several times by the rifle and transferred to antibody pre-coated strips, capped and incubated 4 degrees overnight. The remaining steps are performed with reference to the methods provided in the elisa kit (e.g. as described in Cellsignaling Technology # 7212C).

The results show that the compound of the invention can effectively inhibit TRKA and TRKB phosphorylation levels at a cytological level, and particularly shown in a table 2.

TABLE 2

Compound (I)	TRKA(nm)	TRKB(nm)
Example 2	++	++

Bioassay example 3 KM12-LUC cell proliferation assay

A human colon cancer cell line KM12-LUC (LUC, stably expressing Luciferace) containing a TPM3-NTRK1 fusion gene is used as a model for evaluating the cytological pharmacodynamics of a compound to be tested. The TRK fusion gene in KM12-LUC cells makes the cells independent of stimulation of extracellular growth factors, and can continuously and spontaneously activate downstream signaling pathways MAPK-ERK, PI3K-AKT and other signaling pathways closely related to cell proliferation. Therefore, inhibition of TRK activity in KM12-LUC cells significantly inhibited cell proliferation.

The method comprises the following steps: the first day, cells were seeded in 384-well plates, 2000 cells/well; adding the compounds to be detected with different concentrations on the next day; on the fifth day, cell activity was measured by adding CellTiter-glo (Promega), and the cell proliferation inhibition rate was calculated for 72 hours. Statistical analysis with prism5 and IC of test compound₅₀The value is obtained.

The results show that the compound of the invention can effectively inhibit the proliferation of KM12-LUC cells, and the details are shown in Table 3

TABLE 3

Compound (I)	KM12-LUC(nm)
Example 2	++

Bioassay example 4 mutant TRKA (G595R), TRKA (G667C) and TRKC (G623R) kinase in vitro Activity assay

Experimental Material

Recombinant human TRKA (G595R), TRKA (G667C), and TRKC (G623R) proteins were purchased from SignalChem. HTRF kinEASE TKkit was purchased from CisbioBioassays. The BioTek microplate reader Synergy Neo 2 plate was used.

Experimental methods

Test compounds were diluted in 4-fold concentration gradient to a final concentration of 10 concentrations from 1 μ M to 0.004nM, two duplicate wells per concentration; the content of DMSO in the assay reaction was 1%.

TRKA (G595R) enzyme reaction:

0.12 ng/. mu.l TRKA (G595R) protein kinase, 1. mu.M TK Substrate-biotin polypeptide Substrate, 4.5. mu.M ATP, 1 × enzymic buffer, 5mM MgCl₂1mM DTT. The detection plate is White Proxiplate384-Plus plate (PerkinElmer), the reaction is carried out for 30 minutes at room temperature, and the reaction system is 10 mu l.

TRKA (G667C) enzyme reaction:

0.026 ng/. mu.l TRKA (G667C) protein kinase, 1. mu.M TK Substrate-biotin polypeptide Substrate, 5.5. mu.M ATP, 1 × enzymic buffer, 5mM MgCl₂1mM DTT. The detection plate is White Proxiplate384-Plus plate (PerkinElmer), the reaction is carried out for 30 minutes at room temperature, and the reaction system is 10 mu l.

TRKC (G623R) enzymatic reaction:

1.0 ng/. mu.l TRKC (G623R) protein kinase, 1. mu.M TK Substrate-biotin polypeptide Substrate, 62.9. mu.M ATP, 1 × enzymic buffer, 5mM MgCl₂1mM DTT. The assay plate was a White Proxiplate384-Plus plate (PerkinElmer) and reacted at room temperature for 50 minutes in a 10. mu.l reaction system.

Reaction detection:

add 10. mu.l of assay reagent to the reaction plate, containing SA-XL665 at a final concentration of 0.125. mu.M and 1 XTK-Antibody 5. mu.l, incubated overnight at room temperature, Synergy Neo 2 read plate.

Data analysis

The value of 665/620Ratio minus the value of the enzyme-free negative control well was converted to the percent inhibition (%) (1-Ratio) by the following equation_test/Ratio _max)×100％。Ratio _maxRatio as a positive control without test Compound_testThe values were measured for each concentration of different compounds. IC50(nM) data were obtained by 4-parameter curve fitting, as shown in Table 4.

TABLE 4

Compound (I)	TRKA(G595R)(nm)
Example 1	++
Example 2	++
Example 3	++
Example 4	++
Example 5	+
Example 6	++
Example 7	++
Example 8	+
Example 9	++
Comparative example 1	++
Comparative example 2	++

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

A compound of formula I or a stereoisomer or racemate thereof, or a pharmaceutically acceptable salt thereof:

wherein the content of the first and second substances,

ring a has a structure represented by the following formula:

each X⁴Each independently is C or N;

each X⁵Each independently is CR₇Or N; wherein R is₇Is hydrogen, deuterium, halogen, NH₂Or OH;

R ₁、R ₂taken together with the nitrogen atom to which they are attached to form a substituted or unsubstituted 6-12 membered fused ring (including bicyclic fused ring, tricyclic fused ring), bridged ring or spiro ring; wherein said fused, bridged or spiro ring contains 1 to 3 heteroatoms selected from nitrogen atoms, oxygen atoms and sulfur atoms;

R _Ais composed of

Wherein the content of the first and second substances,

X ₁、X ₂、X ₃each independently is CR₆Or N; r₆Is hydrogen, deuterium, hydroxy, halogen, substituted or unsubstituted C1-C6 alkyl, or substituted or unsubstituted C1-C6 alkoxy;

n is 1,2 or 3;

each R is₃Each independently is hydrogen, deuterium, hydroxy, halogen, trifluoromethyl, cyano, amino, amido, substituted or unsubstituted C1-C6 alkyl, or substituted or unsubstituted C1-C6 alkoxy;

or n is 2 and two R₃co-form-O- (CH)₂) _m-O-; m is an integer of 1 to 4;

R _Bis a substituted or unsubstituted 4-7 membered heterocyclic ring (said heterocyclic ring containing 1 to 3 hetero atoms selected from the group consisting of a nitrogen atom, an oxygen atom and a sulfur atom); - (CH)₂) _m1-、-O-(CH ₂) _m2-O-; wherein m1 and m2 are each independently an integer of 1 to 4;

or R_BIs-substituted or not substitutedsubstituted-C1-C10 alkylene-L₂- (wherein, L)₂Is NH, O, S or S (O)₂) Substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy;

unless otherwise specified, "substituted" means substituted with one or more (e.g., 2, 3,4, etc.) substituents selected from the group consisting of: deuterium, halogen, C1-C6 alkoxy, halogenated C1-C6 alkyl, halogenated C1-C6 alkoxy, halogenated C3-C8 cycloalkyl, methylsulfonyl, -S (═ O)₂NH ₂Oxo (═ O), -CN, hydroxy, -NH₂Carboxy, C1-C6 amido (-C (═ O) -N (Rc)₂or-NH-C (═ O) (Rc), Rc being H or C1-C5 alkyl), C1-C6 alkyl- (C1-C6 amido),
Or a substituted or unsubstituted group selected from: C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 amino, C6-C10 aryl, 5-10 membered heteroaryl having 1-3 heteroatoms selected from N, S and O, 5-12 membered heterocyclyl having 1-3 heteroatoms selected from N, S and O, - (CH) 6 alkyl₂) -C6-C10 aryl, - (CH)₂) - (5-to 10-membered heteroaryl having 1 to 3 heteroatoms selected from N, S and O), and the substituents are selected from the group consisting of: halogen, C1-C6 alkyl, C1-C6 alkoxy, oxo, -CN, -NH₂-OH, C6-C10 aryl, C1-C6 amine, 5-10 membered heteroaryl having 1-3 heteroatoms selected from N, S and O;

the dotted line represents a chemical bond or nothing.
A compound according to claim 1, or a stereoisomer or racemate thereof, or a pharmaceutically acceptable salt thereof: characterized in that ring A is a bicyclic 9-10 membered heteroaromatic ring; wherein the heteroaromatic ring contains 1-3 nitrogen atoms.
A compound according to claim 1, or a stereoisomer or racemate thereof, or a pharmaceutically acceptable salt thereof: its special featureCharacterized in that R is₁、R ₂Together with the nitrogen atom to which they are attached form a substituted or unsubstituted 6-12 membered bicyclic, bridged or spirocyclic ring; the fused ring, bridged ring or spiro ring contains 1-3 nitrogen atoms.
A compound according to claim 1, or a stereoisomer or racemate thereof, or a pharmaceutically acceptable salt thereof: characterized in that R is_ASelected from the group consisting of:

in each formula, X₁、X ₂The definition is the same as the previous definition;

on the aromatic ring, n is 1,2 or 3; and R is₃Is hydrogen, deuterium, hydroxy, halogen, substituted or unsubstituted C1-C6 alkyl, or substituted or unsubstituted C1-C6 alkoxy; or n is 2 and two R₃co-form-O- (CH)₂) _m-O-; m is an integer of 1 to 4;

r in other positions₃、R ₄、R ₅Each independently hydrogen, deuterium, hydroxy, halogen, substituted or unsubstituted C1-C6 alkyl, or substituted or unsubstituted C1-C6 alkoxy.
A compound according to claim 1, or a stereoisomer or racemate thereof, or a pharmaceutically acceptable salt thereof: characterized in that R is_AIs composed of

Wherein, X₁Is CR₆Or N; x₂、X ₃Each independently is CR₆；

n is 1,2 or 3; and R is₃Is hydrogen, deuterium, hydroxy, halogen, substituted or unsubstituted C1-C6 alkyl, or substituted or unsubstituted C1-C6 alkoxy; (ii) a Or n is 2 and two R₃co-form-O- (CH)₂) _m-O-; m is an integer of 1 to 4.
A compound according to claim 1, or a stereoisomer or racemate thereof, or a pharmaceutically acceptable salt thereof: characterized in that it is selected from the following group:
a pharmaceutical composition comprising a therapeutically effective amount of a compound of claim 1, or a stereoisomer or racemate thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.
Use of the compound of claim 1 or a stereoisomer or racemate thereof or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of claim 7, for the preparation of a medicament for the prevention and treatment of a disease associated with abnormal TRK function.
A TRK inhibitor, wherein said inhibitor comprises a compound of claim 1, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate, or solvate thereof.
A method for preparing a compound of claim 1, or a stereoisomer or racemate thereof, or a pharmaceutically acceptable salt thereof, comprising the steps of:

reacting a compound of formula Ia with a compound of formula Ib in an inert solvent, thereby forming a compound of formula I;

wherein R is₁Is C1-C6 alkyl; the remaining groups are as defined in claim 1.